Factor vii composition

ABSTRACT

A stable pharmaceutical composition in liquid form or in solid form, containing factor VII. The composition is free of mannitol, sucrose, and any antioxidant.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending U.S. patentapplication Ser. No. 14/687,499 filed on Apr. 15, 2015; which is acontinuation of U.S. patent application Ser. No. 13/377,680 filed onDec. 12, 2011, now U.S. Pat. No. 9,029,316; which is the National Stageof International Application PCT/FR2010/051229 filed on Jun. 18, 2010;which claims the benefit of French Patent Application 0954390 filed onJun. 26, 2009. The contents of these applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a stable pharmaceutical compositioncomprising factor VII (FVII).

2. Background Information

The coagulation phenomenon comprises a cascade of enzymatic reactionsinvolving coagulation factors of which several are proteases comprisinga serine in their active site. The final step is the conversion ofsoluble fibrinogen into fibrin filaments which surround circulatingcells in their meshes. Coagulation factors are denoted by numbersranging from I to XIII. With the exception of factor XIII, which isinvolved in the final step of coagulation, the other factors areinvolved in reverse order of their numbering; thus, factor XII initiatescoagulation and factor I ends it. Each factor exists in the form of aninactive precursor and in activated form, indicated by the letter a.

Coagulation involves two pathways, one intrinsic, the other extrinsic,resulting in a common final pathway. The combination of the twomechanisms ensures the formation of a solid and flexible blood clot,which withstands blood pressure while at the same time guaranteeingsufficient mobility. Under the action of thrombin, fibrinogen undergoeschemical modifications which result in the formation of fibrin. Fibrinis necessary for clot formation.

The intrinsic pathway comprises the factors present in the circulationand the coagulation process begins right within the blood vessel. Theextrinsic pathway, for its part, involves tissue factors that are notnormally present in the circulation and that are released duringvascular damage. It is when this pathway is activated that a chainreaction occurs, during which an activated coagulation factor triggersthe activation of the subsequent coagulation factor. This pathwayinvolves the intervention of factor VII (FVII) present in the plasma.Activated factor VII, also called proconvertin, is one of the factors,having a molecular weight of approximately 50 kDa, involved in the bloodcoagulation mechanism. It is a glycoprotein of the serine proteasefamily, the synthesis of which in activated form is vitamin K-dependent.In order to initiate the coagulation cascade, FVII must be activated toFVIIa. FVIIa alone (not complexed) has a weak proteolytic activity.Then, once activated, FVIIa complexes with tissue factor (TF), aphospholipid-associated protein, which is released during the vasculardamage. The FVIIa-TF complex subsequently converts factor X to factor Xain the presence of calcium ions. This complex also acts on theactivation of FIX to FIXa, thus catalysing the intrinsic pathway.Factors IXa and Xa in return activate the activated factor VII. FactorXa complexed with activated factor FV and with prothrombinase, convertsprothrombin to thrombin. Thrombin then acts on fibrinogen in order toconvert it to fibrin and also allows the activation of FVIIIa and of FVafrom, respectively, FVIII and FV. Thrombin makes it possible, for itspart, when it is in the presence of calcium, to activate factor XIIIaresponsible for consolidation of the fibrin clot.

Nevertheless, when a coagulation factor is lacking, the cascade ofreactions is interrupted or defective and the term abnormal coagulationis then applied.

Activated factor VII acts locally in the presence of tissue factorreleased after tissue damage causing haemorrhage, even in the absence offactor VIII or IX. For this reason, factor VII, preferably in activatedform, has been used for a long time for the treatment of certain bloodcoagulation disorders manifested by bleeding. The factor is involved inmany pathological conditions, such as haemophilia type A or B, in whichthe patients exhibit factor VIII or IX inhibitors, acquired haemophiliaor congenital factor VII deficiency, and as a product for preventinghaemorrhages that can occur during surgical operations.

At the current time, a medicament available on the market for thetreatment of these patients suffering from haemophilia or fromcongenital factor VII deficiency is known. It is NovoSeven®, authorizedon the European market since 1996 and authorized on the American marketin 1999, produced by the Danish company NovoNordisk. NovoSeven® is amedicament of which the active ingredient is eptacog alfa (recombinanthuman activated coagulation factor VII produced by genetic engineeringfrom BHK baby hamster kidney cells). This product also contains sodiumchloride (2.92 g/l), calcium chloride dihydrate (1.47 g/l),glycylglycine (1.32 g/l), polysorbate 80 (0.07 g/l) and mannitol (30g/l).

A variant of NovoSeven also exists, called NovoSeven® RT, which enablesstorage of the product at room temperature (25° C.). This second productconsists of sodium chloride (2.92 g/l), calcium chloride dihydrate (1.47g/l), glycylglycine (1.32 g/l), polysorbate 80 (0.07 g/l), mannitol (25g/l), and hydrochloric acid and sodium hydroxide for adjusting the pH,and also contains sucrose (10 g/l) and methionine (0.5 g/l) (used asantioxidant). The returning of this product to solution requires waterfor injection, but also histidine. NovoSeven® RT was authorized on theEuropean and American markets in 2008.

The main therapeutic indication for recombinant FVIIa (rFVIIa) concernsthe treatment of spontaneous or surgical bleeding in type Ahaemophiliacs having developed anti-factor VIII antibodies and type Bhaemophiliacs having developed anti-factor IX antibodies. In Europe, itis also indicated for use in patients with a congenital FVII deficiencyand in patients suffering from Glanzmann's thrombasthenia. In addition,numerous publications report the efficiency of rFVIIa in controllinghaemorrhage during surgical procedures, in patients who have neither acongenital deficiency in a coagulation factor nor thrombasthenia.

An article by Nedergaard et al., 2008 [Nedergaard H. et al., In vitrostability of lyophilized and reconstituted recombinant activated factorVII formulated for storage at room temperature, Clinical Therapeutics,Vol 30, No. 7, p 1309-1315, 2008] teaches that NovoSeven® RT remainsstable for a period of 24 months at 25° C., 12 months at 30° C., 6months at 40° C. and 12 hours at 50° C. and 60° C. in its lyophilizedform. Furthermore, this product is stable only for six hours afterliquid reconstitution thereof, and consequently it is recommended toperform the injection within three hours following reconstitution. Thisproduct therefore exhibits, by virtue of this stability, handlingdifficulties and constraints in terms of administration times.

Patent EP 1 210 361 discloses the advantage of using glycylglycine forstabilizing a lyophilized composition of activated factor VII.

Patent application WO 2004/000347 proposes various other stabilizingagents.

The publication by Soenderkaer S. et al., 2004 [Soenderkaer S. et al.,Effects of sucrose on rFVIIa aggregation and methionine oxidation,European Journal of Pharmaceutical Sciences, Vol 21, p 597-606, 2004]describes sucrose as an essential excipient for stabilization againstaggregation and heat denaturation of activated factor VII. According tothis teaching, sucrose enables the protein to retain its native form inaqueous solution, by excluding sugars from the surface of the proteins,which makes it possible to increase the chemical potential of themolecule. Consequently, and by reducing its surface area, the proteinremains in a compact conformation. The presence of sucrose as anexcipient makes it possible to stabilize the activated factor VII in itslyophilized form.

However, the presence of sucrose leads to the induction of antioxidantcompounds in the formulation, resulting in technical and regulatoryconstraints associated with the addition of compounds of this type.

There is therefore today a real need to develop medicaments containingfactor VII which are free of antioxidants, chemically and physicallystable at room temperature, and easy for patients to use, in particularpatients suffering from haemophilia or from congenital factor VIIdeficiency.

SUMMARY OF THE INVENTION

The invention provides a pharmaceutical composition in liquid form or insolid form, comprising factor VII, preferably in the form of factorVIIa, said composition being free of mannitol and of sucrose.

According to one preferred embodiment, the composition is also free ofany antioxidant.

Preferably, the composition comprises at least one hydrophilic aminoacid or amino acid bearing a positively charged side chain, such asarginine.

According to one preferred embodiment, the composition of the inventioncomprises factor VII, at least one hydrophilic amino acid or amino acidbearing a positively charged side chain, at least one hydrophobic aminoacid, and an alkali metal salt, an alkaline-earth metal salt, or a saltof a transition metal, said composition being free of mannitol and ofsucrose.

Advantageously, the composition in solid form can be in the form of apowder or a cake (or plug). The composition preferably has a degree ofmoisture of less than or equal to 3%. According to one particularembodiment, the composition is in lyophilized form.

Another subject of the invention is a method for preparing such acomposition, said method comprising mixing FVII with a buffer solution,adjusting the pH if necessary, and filtering so as to obtain a liquidform. This liquid form can subsequently undergo desiccation in order toobtain the solid form.

The term “buffer solution” includes at least one hydrophilic amino acidor amino acid bearing a positively charged side chain, and an alkalimetal salt, an alkaline-earth metal salt, or a transition metal salt.

Advantageously, the buffer solution also contains at least onehydrophobic amino acid.

Another subject of the invention consists of a method for preparing aninjectable formulation for therapeutic use, said method comprisingdissolving a solid composition as defined herein, in water forinjection.

The invention is also directed towards an injectable formulation thatcan be obtained by means of this method.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention description below refers to the accompanying drawings, ofwhich:

FIG. 1 is a graph which shows that the activity of FVIIa resulting fromthe formulation F3 is preserved after it has been reconstituted inliquid form, following storage for 6 days at 25° C.; and

FIG. 2 is a graph which shows that the formulations F3, F4 and F7 insolid form make it possible to preserve the activity of the protein(FVIIa) (expressed by the FVIIa/FVII:Ag ratio) during storage at 40° C.for 1 month.

DETAILED DESCRIPTION

The applicant provides factor VII compositions which are chemically andphysically stable at room temperature and easy for patients suffering inparticular from haemophilia or from congenital factor VII deficiency touse.

In particular, the novel pharmaceutical composition of the invention hasa stability in solid form of greater than 24 months at a temperatureless than or equal to 25° C.

The composition can therefore be stored at room temperature withoutsubstantial degradation of the factor VII. The term “room temperature”is intended to mean the temperature inside a room, normally includedbetween 10° C. and 30° C., preferably between 15° C. and 25° C.

The term “factor VII” or “FVII” includes polypeptides comprising thesequence 1-406 of wild-type human factor VII (as described in U.S. Pat.No. 4,784,950), or of FVII derived from another species (for example,bovine, porcine, canine, murine). It also comprises the natural allelicvariations of factor VII that can exist, and any form or degree ofglycosylation or other post-translational modification.

The term “factor VII” also includes the FVII variants which have thesame or a greater biological activity compared with the activity of thewild-type form, these variants including in particular the polypeptidesdiffering from wild-type FVIIa by insertion, deletion or substitution ofone or more amino acids.

“Factor VII” or “FVII” comprises uncleaved FVII (zymogen) and activatedfactor VII. Factor VII is used in the composition preferably in itsactivated form.

The term “biological activity of factor VIIa” includes the ability togenerate thrombin, for example at the surface of activated platelets.The activity of factor VII in the composition can be evaluated invarious ways. For example, it can be measured by the ratio between theamount of factor VIIa determined using a coagulation test and the amountof factor VII determined by immunoreactivity with anti-FVII antibodies.

The term “stable composition” signifies herein that the formation ofaggregates (insoluble or soluble) is minimized, and/or that the chemicaldegradation is reduced, the pH is maintained and the conformation of theprotein is not substantially modified during the production or thestorage of the compositions of the invention, such that the biologicalactivity and the stability of the protein are retained. When thecompositions are subjected to lyophilization, the stabilization of thecompositions involves lyoprotection and cryoprotection of the protein.

The term “physical stability” of factor VII refers to the reduction orabsence of formation of insoluble or soluble aggregates of the dimeric,oligomeric or polymeric forms of factor VII, and also to the reductionor absence of any structural denaturation of the molecule.

The term “chemical stability” refers to the reduction or absence of anychemical modification of the factor VII during storage, in the solidstate or in dissolved form, under accelerated conditions. For example,hydrolysis, deamination and/or oxidation phenomena are prevented ordelayed. The oxidation of sulphur-containing amino acids is limited.

For example, the solid compositions of the invention contain lowcontents of oxidized forms and of aggregates at the end of thepreparation method and before storage, for example less than 5% byweight, preferably less than 4%, or else less than 3% or less than 2% byweight of FVII is converted to the oxidized form, and less than 5% byweight, preferably less than 4%, or else less than 3% or less than 2% byweight of FVII is converted to the dimeric or polymeric form. Duringstorage, preferably less than 10% by weight of FVII is converted to theoxidized form, and less than 10% by weight of FVII is converted to thedimeric or polymeric form, after storage at 30° C. after 24 months inthe dark.

A lyophilized composition according to the invention also exhibits astructural stability, i.e. it is capable of forming a cake (or plug)which does not spontaneously crumble, and which is readily soluble inwater before use.

The pharmaceutical composition of the invention is a composition inliquid form or in solid form, comprising factor VII, said compositionbeing free of mannitol and of sucrose.

The content of factor VII, preferably in the form of factor VIIa, in thecomposition of the invention may be the following: between 0.1 and 15mg/ml, preferably between 0.1 and 10 mg/ml, more preferably between 0.2and 5 mg/ml, or else between 0.2 and 2 mg/ml (measured in liquid form,preferably before desiccation, or optionally after reconstitution in theform of an injectable preparation).

Preferably, the composition is free of any sugar, of polyol or ofmethionine.

The sugars to be avoided include, in particular, in addition to sucrose,di- and trisaccharides and polysaccharides, such as dextrose, lactose,maltose, trehalose, cyclodextrins, maltodextrins and dextrans.

The polyols to be avoided include, in particular, in addition tomannitol, sorbitol and xylitol.

More preferably, the composition is free of glycylglycine.

According to one preferred embodiment, the composition of the inventionis also free of any antioxidant. The antioxidants include, for example,one or more of the following compounds: homocysteine, cysteine,cystathionine, methionine, glutathione.

The composition according to the invention comprises at least onehydrophilic amino acid or amino acid bearing a positively charged sidechain, and optionally also at least one hydrophobic amino acid. Thehydrophilic (or polar) amino acids or the amino acids bearing apositively charged side chain include lysine, arginine, histidine,glycine, serine, threonine, tyrosine, asparagine and glutamine.

Among the hydrophilic amino acids or amino acids bearing a positivelycharged side chain, use may preferentially be made of arginine, or oneof its derived salts such as arginine hydrochloride or else argininephosphate.

Amino acids such as glycine and/or lysine, or one of its derived saltssuch as lysine hydrochloride, can be advantageously added.

The addition of a hydrophilic amino acid or amino acid bearing apositively charged side chain, such as arginine, and where appropriateof a hydrophobic amino acid, or even of an alkali metal, alkaline-earthmetal or transition metal salt, promotes the stabilization of factor VIIand the solubilization of the lyophilized forms.

The hydrophobic amino acids (comprising an apolar side chain) include inparticular the following amino acids: alanine, valine, leucine,isoleucine, phenylalanine, tryptophan and proline.

Preferably, the hydrophobic amino acid in the context of this inventionis isoleucine, leucine or a mixture of both.

Preferentially, the composition of the invention comprises an alkalimetal salt, an alkaline-earth metal salt, or a transition metal salt.Mention may in particular be made of trisodium citrate, calcium chlorideor zinc chloride. Preferably, the salt used is preferentially sodiumcitrate or calcium chloride.

Finally, the composition of the invention may comprise one or moredetergents of non-ionic type, such as polysorbates, poloxamers,polyoxyethylene alkyl ethers, a block copolymer ofethylene/polypropylene and polyethylene glycol. Advantageously, thepreferred detergents are polysorbate 80 and polysorbate 20.

In one implementation example, the composition comprises:

-   -   factor VII, preferably in the form of factor VIIa;    -   arginine, optionally in hydrochloride form;    -   isoleucine;    -   lysine;    -   glycine;    -   trisodium citrate or calcium chloride;    -   and, where appropriate, polysorbate 80 or polysorbate 20.

More particularly, the composition may comprise

-   -   factor VII, preferably in the form of factor VIIa;    -   from 10 to 40 g/l of arginine, optionally in hydrochloride form;    -   from 4.2 to 6.6 g/l of isoleucine;    -   from 0.6 to 1.8 g/l of lysine;    -   from 0.6 to 1.8 g/l of glycine;    -   from 1 to 2 g/l of trisodium citrate dihydrate or from 0 to 0.2        g/L of calcium chloride dihydrate;    -   and, where appropriate, from 0 to 0.5 g/l of polysorbate 80.

According to one particular example, the composition comprises

-   -   factor VII (preferably in the form of factor VIIa) at 0.2 to 2        g/l,    -   arginine hydrochloride at 24 g/l,    -   isoleucine at 6 g/l,    -   trisodium citrate dihydrate at 1.5 g/l,    -   glycine at 1.2 g/l,    -   lysine hydrochloride at 1.2 g/l,    -   and/or polysorbate 80 at 0.07 g/l.

According to another particular example, the composition comprises

-   -   factor VII (preferably in the form of factor VIIa) at 0.2 to 2        g/l,    -   arginine hydrochloride at 34 g/l,    -   calcium chloride dihydrate at 0.15 g/l,    -   isoleucine at 6 g/l.

The concentrations are determined with respect to the compositions inliquid form, before desiccation, or after reconstitution in the form ofan injectable preparation.

The Applicant has removed sucrose and also mannitol from its compositionfor pharmaceutical use, these ingredients nevertheless normally beingused as diluents or stabilizers of pharmaceutical formulationscomprising factor VII.

The absence of sucrose and of mannitol offers several advantages.Firstly, the presence of oxidizing components or of endotoxins, whichcan be introduced via the sucrose, is avoided.

It surprisingly emerged that the absence of mannitol allows to preventthe formation of polymorphic forms of mannitol crystals within thecomposition during lyophilization, and limits the risk of impurities,such as in particular the presence of mannose in the composition.

Moreover, against all expectations, the absence of mannitol and ofsucrose from the composition of the invention is not detrimental to thestability of the composition. On the contrary, an increase in the glasstransition temperature of the composition, relative to the availableformulations, for example NovoSeven® RT, is observed.

Glass transition is a second-order transition, i.e. a thermal transitionwhich involves a change in heat capacity, but no latent heat.

It is characteristic of supercooled liquids which are cooled to asufficiently low temperature sufficiently quickly, withoutcrystallizing, and which become a glass, and of amorphous polymers or ofthe amorphous part of crystalline polymers which go from a solid stateto a viscoelastic state. The glass transition temperature or Tg is thetemperature at which this change of state takes place. When a liquidproduct is cooled below this temperature, it becomes solid and brittle,like glass, and is said to be in the glassy state.

Since the mobility of the molecules is blocked when the product is inthe glassy state (only the free-radical functional groups still have alow relative mobility), it is advantageous to store the products attemperatures below their glass transition. A high glass transitiontemperature therefore promotes better stability of the lyophilizedcomposition at high temperatures (>25° C.) and thus decreases thereactivity of the active ingredient [Pikal et al., The Effects ofFormulation Variables on the Stability of Freeze-Dried Human GrowthHormone, Journal of Pharmaceutical Research, Vol 8, p 427-436, 1991].

Furthermore, by virtue of its formulation, the composition forpharmaceutical use according to the invention prevents aggregation ofthe protein.

Indeed, in the presence of mannitol and of sucrose as in the NovoSeven®RT formulation, the glass transition temperature is 45° C. The glasstransition temperature of the composition of the invention, which isfree of sucrose and of mannitol, is above 60° C., and generally between74 and 93° C., which makes it possible to envisage storage outside arefrigerator and even at temperatures above 25° C. (cf. FIG. 2).

The factor VII is generally a human factor VII. It can be obtained invarious ways, for example from the non-cryoprecipitable fraction ofhuman plasma, or by genetic engineering from cells, or else fromtransgenic animals.

Preferably, the factor VII (preferably in the form of factor VIIa) isproduced in particular in the milk of a transgenic animal, theformulation of the invention making it possible for the factor VII toretain a satisfactory biological activity after lyophilization.

In one preferred embodiment, the human factor VII is produced in themilk of nonhuman transgenic mammals genetically modified so as toproduce this protein. Preferably, it is the milk of a transgenic doerabbit or of a transgenic goat.

The secretion of factor VII by the mammary glands, which enables itssecretion in the milk of the transgenic mammal, involves controlling theexpression of the factor VII in a tissue-dependent manner. Such methodsof control are well known to those skilled in the art. The expression iscontrolled by virtue of sequences which allow the expression of theprotein towards a particular tissue of the animal. They are inparticular WAP, beta-casein and beta-lactoglobulin promoter sequencesand signal peptide sequences. The method for extracting proteins ofinterest from the milk of transgenic animals is described in patent EP 0264 166.

The composition of the invention can be obtained using any customarytechnique.

In particular, the composition of the invention can be obtained bycarrying out a method comprising mixing the factor VII with a buffersolution, adjusting the pH if necessary, filtering so as to obtain aliquid form, and then desiccating, if necessary, so as to obtain a solidform.

Preferably, the pH of the solution before desiccation is between 4.0 and9.0, more particularly in the ranges between 4.0 and 8.0; 4.0 and 7.5;4.5 and 7.5; 5.0 and 7.5; 5.5 and 7.0; 6.0 and 7.5; 6.5 and 7.5.

Desiccation is a process for extensive elimination of water. It is adehydration aimed at eliminating as much water as possible. Thisphenomenon may be natural or forced. This desiccation can be carried outby means of lyophilization, spray-drying or cryo-spray-dryingtechniques. The preferred method for obtaining the solid form of thecomposition for pharmaceutical use according to the invention islyophilization.

Lyophilization methods are well known to those skilled in the art, see,for example [Wang et al., Lyophilization and development of solidprotein pharmaceuticals, International Journal of Pharmaceutics, Vol203, p 1-60, 2000].

Other methods suitable for reducing the degree of moisture or the watercontent of the composition can be envisaged. Preferably, the degree ofmoisture is less than or equal to 3% by weight, preferably less than orequal to 2.5%, preferably less than or equal to 2%, preferably less thanor equal to 1.5%.

The composition according to the invention may be advantageouslysubjected to a method for elimination or inactivation of infectiousagents, for example by dry-heating the lyophilisate.

The solid composition according to the invention, preferably inlyophilized form, can be dissolved in water for injection (WFI), so asto obtain a formulation for therapeutic use.

Preferably, the factor VII can be dissolved in pure water, which isadvantageous compared with more complex reconstitution solvents, such asthe solvent with histidine used in the NovoSeven® RT product.

The liquid composition (before desiccation) according to the inventionhas a chemical or physical stability of at least 3 days between 2 and 8°C.

The solid composition according to the invention has a chemical orphysical stability in solid form of greater than 24 months, andgenerally up to at least 36 months, at a temperature less than or equalto 25° C. The reconstituted injectable formulation is also very stable,its chemical and physical stability in liquid form being greater than 6hours at 25° C., preferably greater than 12 hours, preferably greaterthan 24 h, more preferably greater than one week.

The pharmaceutical composition in liquid form or solid form or theinjectable formulation is of use for the treatment of variouspathological conditions.

A subject of the invention is a pharmaceutical composition, orinjectable formulation as defined above, for use in the treatment ofhaemophilia or of congenital factor VII deficiency.

A method for treating haemophilia or congenital factor VII deficiency,in which an effective amount of the injectable formulation described isadministered to a patient requiring such a treatment, is also described.

The haemophilia may be type A or type B. Haemophilia type A ischaracterized by a factor VIII deficiency, whereas haemophilia type B,for its part, is the result of a factor IX deficiency. Congenital factorVII deficiency is a rare hereditary haemorrhagic disease inherited byautosomal recessive transmission, caused by a decrease in or an absenceof coagulation factor VII.

The injectable formulation can be administered parenterally(intravenously, subcutaneously, intramuscularly), in an amount assessedby the practitioner. The administration of the liquid form (beforedesiccation) or of the solid form, via any suitable route and anysuitable means, is not excluded.

The following examples and figures illustrate the invention without,however, limiting the scope thereof.

EXAMPLES

The FVII used in the examples is obtained from the milk of transgenicdoe rabbits, as described in patent application WO 2008099077. It ishuman factor VII which is activated during its purification.

Example 1 Preparation of the Formulations 1.1: Preparation of LiquidFormulations F1 to F7:

Purified and activated factor VII (10-20 ml) at an approximateconcentration of 0.6 mg/ml (formulations F1 to F5) and 0.4 mg/ml(formulations F6 and F7), respectively, was dialysed for 12 hoursagainst 2 litres of buffer solution, as defined in the corresponding rowin Table 1. The pH (6.0±0.2) was adjusted with either 1M NaOH or 1M HCl.The formulated FVIIa solutions were filtered and distributed intobottles in a proportion of 0.5 ml per bottle. The bottles were thenpre-stoppered using bromobutyl stoppers.

1.2: Preparation of Lyophilized Formulations Resulting from the LiquidFormulations F1 to F7 Prepared in Point 1.1 Above:

The bottles were lyophilized according to a predefined cycle. In orderto make it possible to detect the end of the desiccation, thelyophilizer was equipped with a capacitance moisture sensor. At the endof the lyophilization cycle, the bottles were stoppered under vacuum andsealed with aluminium capsules.

1.3: Preparation of the Liquid Formulation F8:

Purified and activated factor VII (0.4 mg/ml) was formulated by bufferexchange on a Superdex 200 gel filtration column. The column wasinitially equilibrated with a buffer solution comprising trisodiumcitrate (1.0 g/l), arginine hydrochloride (30 g/l) and isoleucine (6.0g/l) (see Table: F8). The pH (7.0±0.2) was adjusted. The concentrationof the eluate was determined at approximately 0.4 mg/ml.

The formulated FVIIa solution was filtered and distributed into bottlesin a proportion of 0.5 ml per bottle. The bottles were thenpre-stoppered using bromobutyl stoppers.

1.4: Preparation of a Lyophilized Formulation Resulting from LiquidFormulation F8 Prepared in Point 1.3 Above:

The bottles were lyophilized according to a predefined cycle. In orderto monitor the end of the desiccation, the lyophilizer was equipped witha capacitance moisture sensor. At the end of the lyophilization cycle,the bottles were stoppered under vacuum and sealed with aluminiumcapsules.

1.5: Preparation of the Liquid Formulation F9 and Lyophilization:

Purified and activated factor VII (approximately 0.4 mg/ml) wasformulated by buffer exchange on a Superdex 200 gel filtration column.The column was initially equilibrated with a buffer solution comprisingtrisodium citrate (1.0 g/l), arginine hydrochloride (30 g/l) andisoleucine (6.0 g/l) (see Table: F9). The pH (7.0±0.2) was adjusted.Glycine (1.2 mg/ml) and lysine hydrochloride (1.2 mg/ml) were added.

The formulated FVIIa solution was filtered and distributed into bottlesin a proportion of 1.0 ml per bottle. The bottles were thenpre-stoppered using bromobutyl stoppers.

The bottles were then lyophilized in the same way as previously, andstored at 40° C. for 6 months.

1.6: Preparation of the Liquid Formulations F10 and F11 andLyophilization:

Purified and activated factor VII at an approximate concentration of 1.0mg/ml (F10) and 0.8 mg/ml (F11), respectively, was formulated bydialysis in tubing with the buffer as defined in tables 3 and 4 (seetable: F10 and F11). The pH (7.0±0.2) was adjusted.

The formulated FVIIa solutions were filtered and distributed intobottles in a proportion of 1.0 ml per bottle. The bottles were thenpre-stoppered using bromobutyl stoppers.

The bottles were then lyophilized in the same way as previously.

The formulation F10 was stored under the following conditions:

-   -   liquid at 5° C. for 144 hours then at 25° C. for 6 hours,    -   freezing and thawing cycle (4 consecutive cycles),    -   lyophilized and stored at 25° C. and 40° C. for 6 months,    -   frozen at <−70° C. for 6 months.

The formulation F11 was stored under the following conditions:

-   -   liquid for 72 hours at 5° C. plus a further 6 hours at 25° C.,    -   lyophilized and stored at 25° C. and 40° C.,    -   frozen at <−70° C. in the buffer before the dialysis step:

Trometamol (2.42 mg/ml), NaCl (8.77 mg/ml) and mannitol (30 mg/ml).

Example 2 Formulation Tests Materials and Methods: 2.1. Measurements ofDynamic Light Scattering, Hereinafter “DLS”

500 microlitres of each sample resulting from the lyophilization wereplaced in a microcuvette (Plastibrand®, Wertheim, Germany), which wastransferred into a Zetasizer Nano instrument (Malvern Instruments,Worcestershire, UK). This instrument operates with a 4 mW He—Ne laser at633 nm, and a non-invasive backscatter, or NIBS, technique.

The size distribution of the monomeric populations of the protein byintensity and volume was calculated using the Dispersion TechnologySoftware from Malvern (version 4.00). The refractive indices of thematerial and of the dispersant were defined at 1.33 and 1.45,respectively. The temperature during the measurements was controlled andfixed at 20° C. Quality parameters for the formulations tested includethe diameter of the population of proteins in monomer form and theintensity of scattering of the population of proteins.

2.2. Visual Inspection of the Formulations Reconstituted

The test formulation was reconstituted from the lyophilized formulationwith 0.5 g of water for injection. The solvent was injected into thebottle of lyophilisate using a syringe, via the stopper.

After complete dissolution of the cake, the reconstituted product wasexamined according to the visual inspection method relating to theEuropean Pharmacopoeia (Methods of Analysis—Pharmaceutical TechnicalProcedures—Particulate contamination-visible particles—paragraph2.9.20).

The formulations tested were classified semi-quantitatively, accordingto their degree of contamination with visible particles:

−=no visible particle

ε=some rare visible particles

+=small amount of visible particles

++=large amount of visible particles

+++=very large amount of visible particles

All the tests were carried out independently, and by three differentoperators.

2.3. Filter Test for Aggregate Detection

The method used is taken from the article [Li et al., A simple methodfor the detection of insoluble aggregates in protein formulations,Journal of Pharmaceutical Sciences Vol 96 (7), p 1840-1843, 2007].

A Millex® GV 0.2 μm sterile membrane filter was rinsed with 3 ml ofwater and then the same volume of buffer solution. 0.5 ml of protein(FVIIa) solution to be analysed was subsequently filtered through thisfilter. The filter was then rinsed again with equal volumes of water forinjection (3 ml) and then of buffer solution (3 ml). The proteinfractions retained by the filter were stained by adding 2 ml of stainingsolution (Reversible Protein Detection KIT from SIGMA). The stainingsolution was kept in contact with the filtration membrane for 5 minbefore flow. The membrane was then rinsed again as described above. Theformulations tested were classified semi-quantitatively, according totheir degree of stained particles observed on the filter, originatingfrom proteins:

−=No visible particle detected, originating from proteins

ε=Some rare visible particles detected, originating from proteins

+=Small amount of visible particles detected, originating from proteins

++=Large amount of visible particles detected, originating from proteins

+++=Very large amount of visible particles detected, originating fromproteins.

2.4. Determination of the Glass Transition Temperature (Tg)

The glass transition temperature was determined by means of a DSC 7differential scanning thermoanalyser (Perkin Elmer) calibrated usingindium (melting temperature (Tm) 156.6° C.) and n-octadecane (Tm 38.2°C.). The samples were subjected to temperatures from −50 to 138° C. at aspeed of 20° C./min. Helium was used to carry out the experiments at atemperature below room temperature. The glass transition temperature wastaken at the median point of the endothermic change in the apparentspecific heat. Two measurements were carried out and the mean gives theTg.

2.5. Determination of the FVIIa/FVII:Ag Ratio

The activity of the factor VII in the composition is evaluated by theratio between the amount of factor VIIa determined using a coagulationtest and the amount of factor VII determined by immunoreactivity withanti-FVII antibodies.

Assaying of factor VII (hereinafter denoted “factor VII antigen” or“FVIIAg”):

The FVII is assayed using an immunoenzymatic method (ELISA) withcommercial reagents (Diagnostica Stago). Briefly, the factor VII to beassayed is captured by an anti-human factor VII antibody immobilized ona solid phase. The factor VII attached is then recognized by aperoxidase-conjugated immunoconjugate. The amount of peroxidase bound ismeasured through its activity on the substrate ortho-phenylenediamine inthe presence of aqueous hydrogen peroxide. The intensity of thecoloration, after the reaction has been stopped using a strong acid,depends on the amount of factor VII initially present in the sample.

Assaying of factor FVII in activated form:

The activated factor FVII (FVIIa) is measured using a chronometricmethod with commercial reagents (Diagnostica Stago). Recombinant solubletissue factor (rsTF) has a factor VIIa cofactor function and allowsplasma coagulation in the presence of phospholipids and calcium. In thissystem, the coagulation time obtained will be dependent on the amount offactor VII contained in the test sample. The rsTF does not activatefactor VII to factor VIIa; consequently, the factor VII contained in theplasma does not interfere in the assay.

2.6: Molecular Size Distribution (MSD):

The molecular size distribution is determined by means of achromatography system equipped with a pump, a thermostatic injector, aUV detector, and a computer acquisition system, using a Superdex Tricorn200 10/300 GL column (GE Healthcare, ref. 17-5175-01). The mobile phaseis composed of 0.01M phosphate buffer, 0.138M sodium chloride and0.0027M potassium chloride at pH 7.4. Its flow rate is 0.4 ml/min. Forthe analysis, 100 μl of the sample are injected. The UV detection iscarried out at 280 nm.

2.7: SDS-PAGE (Reduced/Nonreduced):

The quality of the samples in terms of the amount of covalent aggregatesand of fragments was evaluated by analysis of the differences related tothe weight of the proteins, by SDS-PAGE electrophoretic migration, undernonreducing conditions and under reducing conditions on a Novex systemusing gels in 2-(N-morpholino)ethanesulphonic acid or MES buffer(Invitrogen). An amount corresponding to 2 μg of protein was loaded. Theproteins were visualized by Coomassie blue and/or silver nitratestaining.

2.8: IEF

The separation of the various FVII isoforms according to theirisoelectric point was carried out by isoelectric focussing (IEF). Themigration is carried out on a Focugel 3-10 ETC (Gelcompany) on aMultiphor system under native (nonreducing and nondenaturing)conditions. The product was subjected to desalting on a filtration unit(exclusion size 10 kDa). After having determined the concentration ofthe product via the OD at 280 nm, 30 μg were loaded onto the gel. Thecomparison of the populations contained in the product with the twodifferent pI standards (standard pI 5.5-10.5 from GE and standard pI5.4-5.9-6.6 from Sigma, loading of the individual standards) afterstaining with CBB-G250 (Coomassie Brilliant Blue G250) made it possibleto identify the pI of the FVII isoforms (quantification using theQuantity one software, BioRad).

Results: 1. Formulations F1 to F8

In table 1 below, the formulations F1 and F2 are given by way ofcomparison.

Among the compositions according to the invention (F3 to F8), theformulations F3, F4 and F7 are preferred.

According to this table, the formulations F4, F5 and F7 show apercentage intensity of the monomeric population of the protein(respectively 58%, 61% and 55%) which is greater than those obtained inthe NovoSeven® (F1) (9%) and NovoSeven RT® (F2) (27%) formulations.These results reflect an increase in the stability of the product insolid form and the presence of monomers makes it possible to decreasethe risks of immunological reactions.

As regards the FVIIa/FVII:Ag ratio, the formulations F3, F4 and F8exhibit FVIIa/FVII:Ag ratios before lyophilization and afterreconstitution in liquid form that are higher than those obtained withformulations such as those of NovoSeven® (F1) and of NovoSeven RT® (F2),which reflects the fact that the protein (FVIIa) contained in thecomposition according to the invention is more active. An FVIIa/FVII:Agratio before lyophilization of 17 for F3 and 23 for F8 can be observed,compared with 14 for the ratios obtained with the NovoSeven® (F1) andNovoSeven RT® formulations, and an FVIIa/FVII:Ag ratio afterreconstitution in liquid form of 19 for F3 and 20 for F8 can beobserved, compared with 15 for the ratios obtained with the NovoSeven®(F1) and NovoSeven RT® formulations.

As regards the experiments carried out on the glass transitiontemperature (Tg), the formulations F3 to F8 according to the inventionshow an increase in the glass transition temperature. A glass transitiontemperature equal to 75° C. for the formulation F3, and of 93° C. forthe formulation F7, are in particular obtained. These results show thatthe protein (FVIIa) is advantageously stable in these formulations. Thisis because a high Tg allows improved stability of the protein byreducing the mobility and the reactivity of the active ingredient.Furthermore, these results clearly show that the products according tothe invention can be stored outside a refrigerator, contrary to theNovoSeven product from the company NovoNordisk.

As regards the filter test for aggregate detection, the results indicatethat the formulations F3 to F8 according to the invention show fewaggregates, or even a total absence of aggregates, for the formulationsF4, F7 and F8.

As shown in FIG. 1, the activity of the FVII resulting from theformulation F3 (in liquid form after reconstitution) is stillsatisfactory after 6 days, with an activity of at least 80%.

As shown in FIG. 2, the activity of the FVII resulting from theformulation F3 (in solid form) is still satisfactory after one month,with an activity of at least 80% for the formulation F3, and at least90% for the formulation F4.

TABLE 1 Characteristics of the compositions prepared (F1 to F8)(comparative) Visual aspect obtained according to the European Size and% intensity Pharmacopoeia after lyophilization Ratio U/ml method of themonomeric FVIIa/FVII:Ag After population of After Aggre- Beforereconsti- the protein* Before reconsti- gate Formulation Buffer solutionFVIIa con- lyophi- tution in Diameter % inten- lyophi- tution in Tgdetection identification composition centration lization liquid form(nm)* sity* lization liquid form (° C.) test F1 Glycylglycine (1.32 g/l)0.6 mg/ml — — 7 9 14 15 45 ++ Formulation CaCl₂•2H₂O (1.47 g/l) similarto NaCl (2.92 g/l) Novoseven Mannitol (30 g/l) Polysorbate 80 (0.07 g/l)F2 Methionine (0.5 g/l) 0.6 mg/ml — — 6 27 14 15 (Histidine) 41 ++Formulation Glycylglycine (1.32 g/l) 15 (water) similar to CaCl₂•2H₂O(1.47 g/l) Novoseven NaCl (2.92 g/l) RT Sucrose (10 g/l) Mannitol (25g/l) Polysorbate 80 (0.07 g/l) F3 Glycine (1.2 g/l) 0.6 mg/ml — — 7 3917 19 75 + Lysine, HCl (1.2 g/l) Trisodium citrate•2H₂O (1.5 g/l)Isoleucine (6 g/l) Arginine, HCl (24 g/l) F4 Glycine (1.2 g/l) 0.6 mg/ml— — 7 58 16 17 74 ε Lysine, HCl (1.2 g/l) Trisodium citrate•2H₂O (1.5g/l) Isoleucine (6 g/l) Arginine, HCl (24 g/l) Polysorbate 80 (0.07 g/l)F5 Glycine (1.2 g/l) 0.6 mg/ml — — 7 61 14 13 78 ε Lysine, HCl (1.2 g/l)Trisodium citrate•2H₂O (1.5 g/l) Arginine, HCl (30 g/l) Polysorbate 80(0.07 g/l) F6 CaCl₂•2H₂O (0.15 g/l), 0.4 mg/ml — — 7 21 13 13 84 +Arginine, HCl (40 g/l) F7 CaCl₂•2H₂O (0.15 g/l), 0.4 mg/ml — — 7 55 1414 93 ε Arginine,HCl (34 g/1), Isoleucine (6 g/l) F8 Trisodiumcitrate•2H₂O 0.4 mg/ml — — 7 23 23 20 91 ε (1 g/l), Arginine, HCl (30g/l), Isoleucine (6 g/l) *size determined by DLS. The compositions F1and F2 are given by way of comparison and correspond to the NovoSeven ®(F1) and NovoSeven RT ® (F2) formulations.

2. Formulations F9 to F11—Stability Studies

The results of the tests carried out on these formulations are given intables 2 to 4 below.

2.1 Stability Study: Formulation F9

The formulation F9 remains stable also after 6 months at 40° C., inlyophilized form. In addition, the results show that the FVII does notshow any sign of obvious degradation after 6 months at 40° C.

2.2 Stability Study: Formulation F10

The studies carried out on the formulation F10 (Table 3) made itpossible to show that the FVIIa is stable for 48 hours at 5° C., inliquid form.

As can also be seen in Table 2, the formulation F10 remains stable afterfour freezing/thawing cycles and the results obtained attest to anabsence of degradation of the FVII.

The formulation F10 remains stable also after 6 months at 25° C. or at40° C., in lyophilized form. The results of this study show that theFVII has not undergone any degradation.

The formulation F10 (non-lyophilized) remains stable also after 6 monthsfrozen at a temperature below −70° C. The results obtained prove thatthe FVII has not undergone any degradation.

2.3 Stability Study: Formulation F11

The formulation F11 (Table 4) is stable after 6 months at 25° C. or at40° C., in lyophilized form. The results of this study show that theFVII has not undergone any degradation.

The formulation F11 is stable also after 6 months frozen at atemperature below −70° C., in lyophilized form. The results obtainedprove that the FVII has not undergone any degradation.

The study of stability of the formulation F11 in liquid form shows thatthe FVII remains stable during the 72 hours at 5° C. plus the 6 hours at25° C.

TABLE 2 Characteristics of the compositions prepared (F9) (comparative)Visual appearance % intensity of the obtained according populationcontaining to the European the protein in its Aggregate Pharmacopoeiamonomeric form Ratio U/ml detection MSD analyses method (measurement byDLS) FVIIa/FVII:Ag test (% monomers) SDS- Formulation T = 0 (after T = 6T = 0 (after T = 6 T = 0 (after T = 6 T = 6 T = 6 PAGE and F9lyophilization) months lyophilization) months lyophilization) monthsmonths T = 0 months IEF Lyophilized — — 61% 52% 16 16 — 86% 90%Comparable at 40° C. T = 0 and T = 6 months profiles

TABLE 3 Characteristics of the compositions prepared (F10) (comparative)Visual appearance % intensity of the obtained according populationcontaining to the European the protein in its Aggregate SDS- FormulationPharmacopoeia monomeric form Ratio U/ml detection MSD analyses PAGE andF10 method (measurement by DLS) FVIIa/FVII:Ag test (% monomers) IEF T =48 h T = 48 h T = 48 h T = 48 h T = 48 h T = 0 at 5° C. T = 0 at 5° C. T= 0 at 5° C. at 5° C. T = 0 at 5° C. Liquid at — — 73% 70% 16 14 ε 92%88% Comparable 5° C. T = 0 and T = 48 H 5° C. profiles T = 0 (after T =0 (after T = 0 (after lyophi- T = 6 lyophi- T = 6 lyophi- T = 6 T = 6 T= 6 lization) months lization) months lization) months months T = 0months Lyophilized — — 55% 71% 16 16 ε 91% 93% Comparable at 25° C. T =0 and T = 6 months profiles Lyophilized — — 55% 65% 16 15 ε 91% 92%Comparable at 40° C. T = 0 and T = 6 months profiles Freezing — — 73%87% 16 16 ε 92% 92% Comparable at <−70° C. T = 0 and T = 6 monthsprofiles T = 4 T = 4 T = 4 T = 4 T = 4 T = 0 cycles T = 0 cycles T = 0cycles cycles T = 0 cycles 4 freezing/ — — 73% 63% 16 13 ε 92% 89%Comparable thawing T = 0 and T = cycles 4 cycles profiles

TABLE 4 Characteristics of the compositions prepared (F11) (comparative)Visual appearance % intensity of the obtained according populationcontaining to the European the protein in its Aggregate SDS- FormulationPharmacopoeia monomeric form Ratio U/ml detection MSD analyses PAGE andF11 method (measurement by DLS) FVIIa/FVII:Ag test (% monomers) IEF T =72 h T = 72 h T = 72 h T = 72 h at T = 72 h at 5° C. + at 5° C. + at 5°C. + 5° C. + at 5° C. + 6 h at 6 h at 6 h at 6 h at 6 h at T = 0 25° C.T = 0 25° C. T = 0 25° C. 25° C. T = 0 25° C. Liquid at — — 65% 66% 1521 NP NP NP NP 5° C. T = 0 (after T = 0 (after T = 0 (after lyophi- T =6 lyophi- T = 6 lyophi- T = 6 T = 6 T = 6 lization) months lization)months lization) months months T = 0 months Lyophilized — — 65% 66% 1920 ε 98% 93% Comparable at 25° C. T = 0 and T = 6 months profilesLyophilized — — 65% 55% 19 19 ε 98% 97% Comparable at 40° C. T = 0 and T= 6 months profiles Freezing — — 87% 87% 19 18 ε NP 97% Comparable at<−70° C. T = 0 and T = 6 months profiles NP = not performed

1. A pharmaceutical composition in liquid form or in solid form,comprising factor VII, said composition being free of mannitol and ofsucrose.
 2. The pharmaceutical composition of claim 1 in liquid form,comprising factor VII, said composition being free of mannitol and ofsucrose.
 3. The pharmaceutical composition of claim 1 in solid form,preferably lyophilized form, comprising factor VII, said compositionbeing free of mannitol and of sucrose.
 4. The pharmaceutical compositionof claim 1, said composition being free of any sugar, of polyol or ofmethionine.
 5. The pharmaceutical composition of claim 1, saidcomposition further being free of glycylglycine.
 6. The pharmaceuticalcomposition of claim 1, in which the factor VII is in activated form(FVIIa).
 7. The pharmaceutical composition of claim 1, said compositionfurther comprising at least one hydrophilic amino acid or amino acidbearing a positively charged side chain and, optionally, at least onehydrophobic amino acid.
 8. The pharmaceutical composition of claim 1,further comprising an alkali metal salt, an alkaline-earth metal salt,or a transition metal salt.
 9. The pharmaceutical composition of claim1, comprising factor VII, preferably in the form of factor VIIa;arginine, optionally in hydrochloride form; isoleucine; lysine,optionally in hydrochloride form; glycine; trisodium citrate or calciumchloride; and, where appropriate, polysorbate 80 or polysorbate
 20. 10.The pharmaceutical composition of claim 9, comprising factor VII,preferably in the form of factor VIIa; from 10 to 40 g/l of arginine,optionally in hydrochloride form; from 4.2 to 6.6 g/l of isoleucine;from 0.6 to 1.8 g/l of lysine, optionally in hydrochloride form; from0.6 to 1.8 g/l of glycine; from 1 to 2 g/l of trisodium citratedihydrate or from 0 to 0.2 g/L of calcium chloride dihydrate; and, whereappropriate, from 0 to 0.5 g/l of polysorbate
 80. 11. The pharmaceuticalcomposition of claim 10, comprising factor VII, preferably in the formof factor Vila, at 0.2 to 2 g/l; arginine hydrochloride at 24 g/l;isoleucine at 6 g/l; trisodium citrate dihydrate at 1.5 g/l; glycine at1.2 g/l; lysine hydrochloride at 1.2 g/l; and/or polysorbate 80 at 0.07g/l.
 12. The pharmaceutical composition of claim 9, comprising factorVII, preferably in the form of factor VIIa, at 0.2 to 2 g/l; argininehydrochloride at 34 g/l; calcium chloride dihydrate at 0.15 g/l; andisoleucine at 6 g/l.
 13. A lyophilized solid composition which can beobtained from a pharmaceutical composition of claim
 1. 14. A method forpreparing a pharmaceutical composition of claim 1, said methodcomprising mixing the factor VII with a buffer solution, adjusting thepH if necessary, filtering, and then desiccating if necessary so as toobtain the solid form.
 15. A method for preparing an injectableformulation for therapeutic use, said method comprising dissolving asolid composition, as defined in claim 1, in water for injection.
 16. Aninjectable formulation prepared by the method of claim
 15. 17. A methodfor treating haemophilia or congenital factor VII deficiency, the methodcomprising administering a pharmaceutical composition as defined inclaim 1 to a patient in need thereof.
 18. A method for treatinghaemophilia or congenital factor VII deficiency, the method comprisingadministering an injectable formulation as defined in claim 16 to apatient in need thereof.
 19. The pharmaceutical composition of claim 2,wherein said composition is free of any sugar, of polyol, or ofmethionine.
 20. The pharmaceutical composition of claim 3, wherein saidcomposition is free of any sugar, of polyol, or of methionine.